Lethal arrhythmias commonly occur after myocardial ischemia, especially when ischemic myocardium is reperfused. These arrhythmias are usually initiated by ectopic activity triggered by early and delayed after depolarizations (EADs and DADs) of the membrane potential. One consequence of ischemia and reperfusion is a rapid migration of polymorphonuclear leukocytes (PMNL) into the infarcted zone. Activated PMNL bind to activated myocytes and release several substances, including oxygen radicals, proteolytic enzymes and inflammatory lipids that increase the extent of myocardial injury (Lucchesi B R, and Mullane K M. (1986) Annu Rev Pharmacol Toxicol 26: 201-224). Depletion of circulating neutrophils or treatment with anti-inflammatory drugs effectively limits the size of the infarct zone and the extent of the damage in hearts from several species (Lucchesi B R, and Mullane K M. (1986) Annu Rev Pharmacol Toxicol 26: 201-224, Mullane K M et al. (1984) J. Pharmacol. Exp. Ther. 228: 510-522, Romson J L et al. (1983) Circulation 67: 1016-1023).
Hoffman et al. (1997, J Cardiovasc Electrophysiol 8:679-687; 1996, J Cardiovasc Electrophysiol 7:120-133) demonstrated that activation of PMNL bound to isolated canine myocytes dramatically changed the myocyte transmembrane action potential. These changes included prolongation of the action potential duration (APD), EADs and in some cases arrest during the plateau phase of the action potential. It was also shown that direct superfusion of myocytes with the inflammatory phospholipid, platelet-activating factor (PAF) mimicked the action of activated PMNL, and that under similar conditions PMNL produce significant levels of PAF. Furthermore, incubation of myocytes with the PAF receptor (PAFR) antagonist, CV-6209, prevented both PAF- and PMNL-induced changes in myocyte membrane potential. PAF also induces arrhythmias in mice that overexpress the PAFR when the lipid is administered at intravenous doses that have little effect on wild-type animals (Ishii S et al. (1997) EMBO J. 16: 133-142). These observations suggested that PMNL-derived PAF could induce triggered activity and thus ventricular arrhythmias.
There is considerable confusion regarding the molecular mechanisms by which PAF could alter the electrical activity of the heart. Although PAF binds to a cell-surface, G-protein-linked receptor and ultimately increases cytosolic Ca2+ levels (Massey C V et al.(1991) J Clin Invest 88: 2106-2116; Montrucchio G et al. (2000) Physiol Rev 80: 1669-1699) little is known about PAF effects on membrane channels. Wahler et al. showed that subnanomolar concentrations of PAF markedly decreased the inwardly rectifying potassium channel IK1 in guinea pig ventricular myocytes (Wahler G M et al. (1990) Mol Cell Biochem 93: 69-76), while Hoffman et al. suggested that depolarizing Na+ current may play a role in the arrhythmogenic action of PAF (Hoffman, B F et al. (1996) J Cardiovasc Electrophysiol 7:120-133).
Here, employing genetically modified mice in which PAFR have been knocked out (Ishii S et al. (1998) T, J Exp Med 187: 1779-1788), the ability of carbanyl-PAF (C-PAF), a non-metabolizable PAF analogue, to alter the membrane potential of isolated murine ventricular myocytes has been tested with the intent of clarifying the mechanisms determining the arrhythmogenic effects of this lipid. It is disclosed here that PAF-mediated cardiac electrophysiologic effects are linked to inhibition of the two-pore domain K+ channel, TASK-1.
In addition, the molecular mechanism of the C-PAF effect on TASK-1 current is elucidated by identifying the epsilon isoform of PKC (PKCε) as a critical component in PAFR signaling. Furthermore, using site-directed mutagenesis, the critical residue that is the target for PKC in the murine and human channels is identified. Finally, data is presented here showing that the phosphorylation-dependent disruption of TASK-1 current also occurs in a rapid-pacing model of atrial fibrillation and in peri-operative atrial fibrillation.